MAVS Protein Is Attenuated by Rotavirus NonstructuralProtein 1Satabdi Nandi, Shampa Chanda, Parikshit Bagchi, Mukti Kant Nayak, Rahul Bhowmick,

Mamta Chawla-Sarkar*

Division of Virology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, West Bengal, India

Abstract

Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. Indeveloping countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishesan intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Amongvarious pattern recognition receptors (PAMPs) of the host, the cytosolic RNA helicases interact with viral RNA to activate theMitochondrial Antiviral Signaling protein (MAVS), which regulates cellular interferon response. With an aim to identify therole of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independentmanner. Rotavirus non-structural protein 1 (NSP1) which is a known IFN antagonist, interacted with MAVS and degraded itin a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of ourknowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. Inaddition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signalingcascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virusorchestrates the cellular antiviral response to its own benefit by various back up strategies.

Citation: Nandi S, Chanda S, Bagchi P, Nayak MK, Bhowmick R, et al. (2014) MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1. PLoS ONE 9(3):e92126. doi:10.1371/journal.pone.0092126

Editor: David A. Leib, Geisel School of Medicine at Dartmouth, United States of America

Received December 11, 2013; Accepted February 17, 2014; Published March 18, 2014

Copyright: � 2014 Nandi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The study was financially supported by Indian Council of Medical Research (ICMR), India and Japan Initiative for Global Research Network on InfectiousDiseases (J-GRID) of the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: chawlam70@gmail.com

Introduction

In response to viral infection there are several pattern

recognition receptors (PRRs) such as the Toll-like receptor

(TLR), Nod-like receptor (NLR), RIG-I-like receptor (RLR) along

with the DNA sensors, which plays significant role in activation of

cellular innate immune response [1]. Among RLRs, Retinoic

Acid-Inducible Gene I (RIG-I) and Melanoma Differentiation-

Associated protein 5 (MDA-5) are the cytosolic receptors which

discriminate between various classes of RNA and DNA viruses in

order to activate interferons (IFNs). RNA viruses can be sensed by

MDA-5 (Picornaviruses), by RIG-I (Hepatitis C virus, Influenza

virus, Newcastle disease virus, Sendai virus, Rabies virus,

Reovirus, Vesicular stomatitis virus and Japanese encephalitis

virus), or by both RIG-I and MDA-5 (Dengue and West Nile virus)

[2,3]. In addition, RIG-I recognizes dsRNA and 59-triphosphate

moiety, whereas the length of dsRNA determines the utilization of

RIG-I and MDA-5 for recognition [4]. After binding with viral

RNA, caspase activation and recruitment domain (CARD) of

RIG-I and MDA-5 interacts with the CARD domain of a

common adaptor protein; mitochondrial antiviral signaling

protein (MAVS: also known as IPS-1/VISA/Cardif). This

interaction leads to the formation of prion-like aggregates of

MAVS CARD domain which signals IKKs and TBK1 for the

activation of IRF3 and NF-kB pathways [5]. A highly synchro-

nized activation of NF-kB and IRF-3 pathways lead to the

assembly of an activating complex comprising various proteins

that drive expression of IFN-b and other IFN mediated antiviral

immunity [6–10]. In order to counteract this antiviral milieu,

viruses have developed various strategies to inhibit the IFN-bsecretion. Interestingly in different classes of virus, the MAVS

protein is abrogated from functioning in order to handicap the

primary innate immune response. In hepatitis B, hepatitis C and

Coxsackievirus B virus, MAVS is cleaved from its mitochondrial

membrane location making it ineffective for downstream signaling

[11–14]. The PB1-F2 protein inhibits MAVS-mediated IFN

synthesis by decreasing the mitochondrial membrane potential

during influenza virus infection [15,16]. During rotavirus (RV)

infection, RIG-I/MDA-5-MAVS pathway leads to the up

regulation of type I IFNs rather than TLR3/TRIF or PKR

pathway [17]. As the virus enters in the host cell, that activation of

the antiviral response by RV is dependent on MAVS/IPS-1 and

IRF3 involving both RIG-I and MDA-5, however IFN-b secretion

during RV infection is regulated by PKR (Protein kinase R)

phosphorylation [18,19]. The central role of MAVS protein

during RV infection was shown by Sen et al where both

transcriptional responses and IFN-b secretion were completely

abrogated in MAVS2/2 MEFs (mouse embryonic fibroblasts)

[18].

Rotaviruses are members of Reoviridae family and are the

single most important etiologic agent of severe infantile (,5 years)

non-bacterial diarrhoea in humans worldwide [20]. It is a non-

enveloped icosahedral virus with 11 double stranded RNA

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segments. Each RNA segment encodes a functional protein except

segment 11 which encodes two nonstructural proteins in +1 open

reading frame (ORF) [21]. Therefore the virus encodes six

structural (VP1-4, VP6-7) and six non structural proteins (NSP1-6)

[20]. Non-structural protein 1 (NSP1) of RV is a 55 KD protein

which plays a vital role in antagonizing the IFN immune response

[22-26,44]. NSP1 is also found to activate PI3K/AKT mediated

anti-apoptotic pathway [27] through its ability to bind p85 subunit

of PI3K for activation of AKT [28], resulting in efficient virus

infection and replication. In addition, NSP1 has shown to

downregulate p53 and TRAF2 (TNF receptor associated factor

2) proteins [29,30]. Except RIG-I, NSP1 mediated degradation of

the above said proteins are proteasome dependent. Thus, there are

many circumstantial evidences suggesting a putative ubiquitin

ligase property of NSP1 [31]. The amino terminus of NSP1 forms

one or two zinc fingers, which contains a highly putative RING-E3

ubiquitin ligase domain [32]. The C-terminal domain of NSP1 is

involved in IRF3 binding [33]. It was shown by Barro et al that

wild-type NSP1, not the C-truncated form, is an antagonist of the

IFN-signaling pathway [24]. It was also shown that RV NSP1

mediates degradation of IFN regulatory factors through targeting

of the dimerization domain [34].

Previous studies have suggested the role of RIG-I/MDA5-

MAVS signaling during RV infection. The critical role of MAVS

in activating early antiviral transcriptional responses is validated in

MAVS2/2 MEFs [17,18]. Therefore the aim of the study was to

know whether RV modulates RIG-I/MDA-5-MAVS pathway by

directly affecting this protein. NSP1 has been shown to degrade

RIG-I [26] but in absence of RIG-I, MDA5 can complement it

and activate IFN through MAVS. Herein, results revealed that

RV protein NSP1 also down regulates the adapter protein MAVS

during RV infection when host PRR mediated IFN-b activation is

critical. Importantly it was found that the degradation was RV

strain independent in nature unlike IRF3 degradation. Until now

NSP1 from different RV strains were found to target different

regulatory factors for antagonizing the IFN response, but this is the

first report where NSP1 is found to target the central protein

MAVS unanimously in a strain independent manner. Although,

the down regulation of IRFs can also serve the same function, the

following finding suggests a backup strategy undertaken by the

same viral protein for efficient IFN down regulation, in case IRFs

are not completely degraded. The study highlights the multistep

control of host innate immunity by a viral protein.

Materials and Methods

Reagents and antibodiesProteasomal inhibitor MG132 was purchased from Sigma–

Aldrich (St. Louis, MO, USA) which was used at 20 mM/ml final

concentration. Poly dA dT (deoxyadenylic-deoxythymidylic) and

poly IC (inosinic-cytidylic acid) were purchased from Invivogen

(San Diego, CA, USA). Both were transfected in cells at a final

concentration of 2 mg/ml and 10 mg/ml respectively. Rabbit

polyclonal antibodies against SA11 NSP1 (480–497 amino acids)

and NSP3 (full length) were gifted by Professor Taniguchi,

Department of Virology and Parasitology, Fujita Health Univer-

sity School of Medicine, Aichi, Japan. Antibody against MAVS

(06-1096) was purchased from Millipore (Billerica, MA). TBK1

(sc-9085) and His probe (sc-803), were from SantaCruz Biotech-

nology (CA, USA). Antibodies against GAPDH (2118), b-Actin

(4967), CoxIV (4844), IRF3 (4302), Phospho-IRF-3 Ser396 (4947),

NF-kB (3017), Lamin A/C (2032), pIkba (2859) and Ikba (9242)

were from Cell Signaling Technology. Antibody against FLAG

epitope (SAB4200071) was from Sigma. All antibodies were used

at 1:1000 dilutions except NSP1 and NSP3 (1:3000).

Cell culture and virus infectionHuman intestinal epithelial (HT29) and human embryonic

kidney epithelial (HEK293) cell lines were cultured in Dulbecco9s

modified Eagle9s medium (DMEM) supplemented with 10% fetal

bovine serum and 1% antibiotic–antimycotic solution (Invitrogen,

Carlsbad, CA). Cells were maintained in 5% CO2 at 37uChumidified incubator. Various rotaviral strains used for the study

were propagated and titers were determined with MA104 cells.

For infection, viruses were activated with acetylated trypsin

(10 mg/ml) at 37uC for 30 min and added to the cells for

adsorption (45 min), followed by washing with media to remove

unbound virus. Infection was continued in fresh medium. Except

for experiments with HEK293 cells, an MOI of 1 was used for

MA104 or HT29 cells in the study. The time of virus removal was

taken as 0 hour post infection (hpi) for all experiments. The end

point in experiments was determined based on time required by

virus to complete its replication in cells resulting in an .80%

cytopathic effect. The end point for HT29 cells varied from 18 to

24 h.

Plasmid constructionVectors expressing human IRF3-5D, human TBK1 and

pTATA-luc-4x-IRF-3 are generous gifts from Dr. Aldofo Gar-

cia-Sastre ( Mount Sinai School of Medicine), Dr Harry Green-

berg (Stanford University) and Dr Stephan Ludwig (University of

Munster) respectively All primers used in the study for respective

constructs are given in Table1. Full length NSP1, NSP2, NSP3,

NSP4, NSP5 and ubiquitin were cloned in pcDNA6 vector

(Invitrogen) with C-terminal His-Tag as described previously [29].

To prepare pcDNA vectors containing the NSP1 ORFs of RV

strains OSU (accession number D38153), DS-1 (accession number

EF672578), Wa (accession number L18943), KU (accession

number AB022769) and different truncated mutants of NSP1,

viral RNA was extracted from infected cells by using TRIzol LS

reagent (Invitrogen). Gene 5 cDNAs were prepared from the RNA

by reverse transcription (RT)-PCR followed by PCR with

respective primer sets. Full length MAVS (accession number

BC044952), region specific mutants of MAVS were cloned in

pFLAG-CMV (Sigma, MO) vector using specific primers.

Quantitative real-time RT-PCRTotal RNA was isolated using TRIzol (Invitrogen, Grand

Island, USA) according to the manufacturer’s instructions. cDNA

was prepared from 1–2 mg of RNA using the Superscript II reverse

transcriptase (Invitrogen) with random hexamer primers. Real-

time PCR reactions (50uC for 2min, 95uC for 10 min, followed by

40 cycles of 95uC for 15 s and 60uC for 30 s and 72uC for 10 min)

were performed in triplicate using SYBR Green (Applied

Biosystems, Foster City, CA, USA) with primers specific for

GAPDH (F-59-AATCCCATCACCATCTTCCAG-39 and R-59-

AAATGAGCCCCAGCCTTC-39), nsp4 (F-59-GTGCAAACGA-

CAGGTGAAATAG-39 and R-59-AGTCACTTCTCTTGGTT-

CATAAGG -39), ISG56 (F-59-TGGGCCTTGCTGAAGT-39

and R-59-GGCCCATCCTTCCTCA-39), MAVS TaqMan probe

ID, IFN-b TaqMan probe ID Hs01077958_s1 were used. Relative

gene expressions were normalized to GAPDH using the formula

22DDCT (DDCT =DCTsample-DCT untreated control).

MAVS Protein Is Attenuated by RV NSP1

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Gel electrophoresis and immunoblot analysesWhole cell lysates was prepared with Totex buffer (20 mM

Hepes at pH 7.9, 0.35 M NaCl, 20% glycerol, 1% NP-40, 1 mM

MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 50 mM NaF and

0.3 mM Na3VO4) containing mixture of protease and

phosphatase inhibitors (Sigma, St. Louis, MO), Samples were

incubated in protein sample buffer (final concentration: 50 mM

Tris, pH 6.8, 1% SDS, 10% glycerol, 1% b-mercaptoethanol, and

0.01% bromphenol blue) for 30 min at either 4 uC or,

alternatively, boiled for 5 min before running SDS-PAGE at

room temperature followed by immunoblotting with specific

antibodies. Primary antibodies were identified with HRP conju-

gated secondary antibody (Pierce, Rockford, IL) and chemilumi-

nescent substrate (Millipore, Billerica, MA). To confirm protein

loading blots were reprobed with GAPDH. The immunoblots

shown are representative of three independent experiments. Blots

were scanned and quantitated using GelDoc XR system and

Quantity One software version 4.6.3 (BioRad, Hercules, CA).

Nuclear protein extractionNuclear and cytosolic protein extracts were prepared with

ProteoJET Cytoplasmic and Nuclear Protein Extraction Kit

(Fermentas Life Science, USA) for NFkB p52 and IRF3

translocation studies.

ImmunoprecipitationCell lysate from infected HT29 cells or transfected HEK293

cells were clarified followed by incubation with specific antibodies

overnight at 4uC and with protein A-Sepharose beads (GE

Healthcare, Sweden), for 4h. Beads were washed 5 times with 1 ml

wash buffer (200 mM Tris pH-8.0, 100 mM NaCl and 0.5% NP-

40) and bound proteins were eluted by boiling for 5 min with SDS

sample buffer before separation on 12% SDS-PAGE gels followed

by immunoblotting with specific antibodies.

Transient transfections and reporter gene assaysHEK293 cells were transfected with Lipofectamine 2000

(Invitrogen) according to the manufacturer’s instructions. IRF3

luciferase reporter gene assays were performed by transfection of

constructs pCMV-MAVS, pTATA-luc-4x-IRF-3 (0.15 mg per 24-

well), which contains the luciferase reporter gene under the control

of four copies of the IRF-3 binding PRDI/III motif of the IFN-bpromoter [35] along with presence or absence of pcD-NSP1. For

dual luciferase NFkB reporter assay, HEK293 cells were

cotransfected with NFkB–luc (containing IL8 promoter), pRL-

TK, pCMV-MAVS and pcD-NSP1 or pcDNA. 24 hour post

transfection, the luciferase activity was measured according to the

manufacturer’s protocol (Promega) using a luminometer (Var-

ioskan multimode reader; Thermo Fisher).

SDD-AGE (semidenaturing detergent agarose gelelectrophoresis)

Semidenaturing detergent agarose gel electrophoresis (SDD-

AGE) was performed according to the protocol followed by Hou et

al [5]. In brief, crude mitochondria from HEK293 cells were

isolated in Buffer A (10 mM Tris-HCl [pH 7.5], 10 mM KCl,

1.5 mM MgCl2, 0.25 M D-mannitol, and Roche EDTA-free

protease inhibitor cocktail) and then resuspended in 1x sample

buffer without b-mercaptoethanol (0.56 TBE, 10% glycerol, 2%

SDS, and 0.0025% bromophenol blue). Mitochondrial pellets

were run on 1.5% agarose gel (Bio-Rad) in the running buffer (1 x

TBE and 0.1% SDS) for 3–4 hours with a constant voltage of

50 V at 4uC. The proteins were transferred to nitrocellulose

membrane by capillary method for 6 hours (RT) followed by

immunoblotting with MAVS Antibody.

Table 1. List of primers designed and used in the study.

NAME SEQUENCE VECTOR

SA11 H96 F KpnI 5’GGT ACC ATG GCT ACT TTT AAA GAT GCA TGC TTT CAT pCDNA6B

SA11 H96 R XhoI 5’CTC GAG TTC TCA TTG TCA TCT TCT GAG TTG GAG A pCDNA6B

OSU F KpnI 5’GGT ACC ATG GCT ACT TTT AAG GAT GCT TGC TAT T pCDNA6B

OSU R XhoI 5’CTC GAG TTT TCA ACA TCA GAT ATA CCG GAA TCA TAG pCDNA6B

DS-1 F KpnI 5’GGT ACC ATG GCT ACT TTT AAA GAT GCT TGC TAT C pCDNA6B

DS-1 R XhoI 5’CTC GAG TTT TCA ATA TCG GAT ATA CCT GAA TCA TGT pCDNA6B

Wa F KpnI 5’GGT ACC ATG GCT ACT TTT AAA GAC GCT TGT TAT TAT pCDNA6B

Wa R XhoI 5’CTC GAG TTT TCA ACA TCA GAT ATA CCA GAA TCA TAT pCDNA6B

KU F KpnI 5’GGT ACC ATG GCT ACT TTT AAA GAT GCT TGT TAT C pCDNA6B

OSU R XhoI 5’CTC GAG TTT TCA ACA TCA GAT ATG CCA GAA TCA TC pCDNA6B

NSP1-N-100 R XhoI 5’CTC GAG TTA ATT GGA TGT TTC ACA GTT CTA AGC pCDNA6B

NSP1-C-395 F KpnI 5’GGT ACC ATG ACC AAA GAC AAA TTA CAG TGT ATC pCDNA6B

NSP1-del IRF3 BD Xho1 5’TCT ACT CGA GCG CCA TTT ACA GTA CTG GAT ATC G pCDNA6B

MAVS F EcoRV 5’G ATA TCA TAT GCC GTT TGC TGA AGA CAA GAC C pCMVFLAG6b

MAVS R KpnI 5’G GTA CCA CTA GTG CAG ACG CCG CCG GTA CAG pCMVFLAG6b

MAVS-CARD R KpnI 5’G GTA CCC TAC GAG GTC CGA GGC TGG TAG CTC TC pCMVFLAG6b

MAVS-CARD-TM F 5’CCA GGG CCC CCC GGG GCT CTG TGG CTC

MAVS-CARD-TM R 5’GGA GCC ACA GAG CCC CGG GGG GCC CTG GCC TCT CAG

Pro-MAVS F EcoRV 5’G ATA TCA GAC CGT CCC CCA GAC CCA CTG pCMVFLAG6b

doi:10.1371/journal.pone.0092126.t001

MAVS Protein Is Attenuated by RV NSP1

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Statistical analysisData are expressed as mean6standard deviations of at least

three independent experiments (n$3). Results from all studies

were compared with unpaired two-tailed Student’s t test and

p,0.05 was considered statistically significant.

Results

Degradation of MAVS during Rotavirus infectionThe expression of MAVS was analysed during the time course

of RV infection in HT 29 cells infected with SA11 (1 M.O.I.)

followed by immunoblot analysis of the cell lysates with MAVS

specific antibody. Immunoblotting revealed degradation of MAVS

protein after infection in a time dependent manner (Figure 1A). To

assess whether RV mediated regulation of MAVS is transcrip-

tional or post transcriptional, transcript were quantitated by

realtime PCR in SA11 infected HT29 cells (0–12 hpi). There was

no significant change of MAVS mRNA (Figure 1B) and thus the

degradation of MAVS after infection was confirmed as a post-

transcriptional phenomenon. To elucidate whether rotaviral entry

or replication is responsible for MAVS degradation, HT 29 cells

were infected with either normal or psoralane-UV-irradiated

replication deficient virus [36]. The extent of virus replication

inability was confirmed by estimating viral titer and quantitating

viral transcript by qRT-PCR (Figure S1). As shown in Figure 1C,

in spite of being structurally and immunologically competent, UV

Figure 1. MAVS is degraded during rotavirus infection. A) HT29 cells were infected with RV strain SA11 (1 M.O.I.) and cell lysates wereprepared at indicated time points. Proteins were separated on 12.5% SDS-PAGE and immunoblotted using MAVS Ab. Membranes were reprobed withNSP1 NSP3, IRF3, and GAPDH antibodies as internal control. Band intensities of MAVS and NSP1 were normalized to the loading control GAPDH andexpressed as a percentage of the protein in mock infected cells. B) HT29 cells were infected with SA11 at (1 M.O.I.) for indicated time points. RNA wasisolated and the nsp4 and mavs transcripts were analyzed by qRT-PCR. Fold changes were obtained by normalizing relative gene expressions togapdh using the formula 22DDCT (DDCT =DCTSample-DCTUntreated control). C) Expression of MAVS in HT29 cells infected with normal or psoralane-UV-irradiated replication deficient RV SA11 simian strain. Band intensities of MAVS and NSP1 were expressed in percentage. D) HEK293 cells weretransfected with pcDNA constructs of NSP5, NSP4, NSP3, NSP2 and NSP1. The expression of MAVS protein was analyzed and expression of viral NSPswere confirmed by immunoblotting using His Ab. E) Dose-dependent degradation of MAVS protein in total cell extracts of HEK293 cells transfectedwith increasing concentration of pcD-NSP1 for 24 hours. The data represent the means 6 the standard deviations (SD) of three independentexperiments.doi:10.1371/journal.pone.0092126.g001

MAVS Protein Is Attenuated by RV NSP1

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treated virus could not degrade MAVS suggesting that viral

replication and synthesis of viral nonstructural proteins plays a role

in the process. Furthermore, when HEK293 cells were transfected

with constructs encoding NSPs (NSP1-NSP5) followed by immu-

noblotting after 24 hours. MAVS degradation was observed only

in NSP1 over expressing cells (Figure 1D). To confirm the loss of

MAVS in presence of NSP1, total cell lysates were prepared, 24h

after transfection of HEK293 cells with increasing concentration

of pcD-NSP1. Immunoblotting revealed degradation of MAVS

with increasing concentration of pcD-NSP1 (Figure 1E).

Rotavirus strain independent degradation of MAVSPrevious studies have shown diverse IFN antagonist activities

mediated by NSP1 of different RV strains [37].Thus, MAVS

degradation was analyzed in HT29 cells following infection with

several common laboratory strains of RV namely porcine OSU,

rhesus RRV, bovine A5-13 and A5-16, human Wa, KU and DS-1

(1 M.O.I.). Cell lysates were immunoblotted with MAVS specific

antibody. As an internal control IRF3 degradation was also

analyzed in same blots. Blots were reprobed with NSP3 antibody

to confirm the infection and replication of RV strains in HT29

cells. As shown in Figure 2, except for A5-16 where a truncated

NSP1 is synthesized, infection with all other strains abrogated

MAVS by almost 50% compared to the mock infected control,

whereas IRF3 degradation was observed only in A5-13, EW and

RRV strains. No IRF3 degradation was observed in OSU, Wa,

KU or DS-1 infected cells. This suggests that MAVS degradation

is independent of RV strain as well as its ability to degrade IRF3.

Furthermore, nsp1 gene from four RV strains was cloned in

pcDNA vector and transiently transfected in HEK293 cells. After

24 hours, cell lysates were immunoblotted for MAVS and IRF3

expression. Consistent with the previous results with RV strains,

degradation of MAVS was observed in cells expressing NSP1

whereas no significant effect on IRF3 was observed. Overall results

suggested that MAVS is an important target during RV infection.

Figure 2. Degradation of MAVS in RV infected HT29 cells and NSP1 transfected HEK293 cells. A, B, C and D) HT29 cells were infected withthe indicated strains of RV (1 M.O.I.) and harvested at various time points. Total cellular protein was harvested and separated by SDS-PAGE.Immunoblot analyses were performed with antibodies to the cellular proteins MAVS, IRF3, GAPDH and the viral proteins NSP1 and NSP3. PorcineOSU, bovine A5-13, A5-16 and human Wa were harvested at 2, 4, 8 and 12 hpi. Murine EW, rhesus RRV, human KU and DS-1 were lysed at 6 and 12hpi.Band intensities of MAVS and IRF3 were normalized to the loading control GAPDH and were expressed as a percentage of the protein in mockinfected cells. The data represent the means 6 SD of three independent experiments. E) Effects of NSP1 proteins of different RV strains on humanMAVS. HEK293 cells were transfected with vectors encoding indicated RV NSP1 proteins and harvested at 24 h p.i. Proteins in cell lysates wereresolved by SDS-PAGE and analyzed by immunoblotting for MAVS, IRF3 and Anti-His antibody.doi:10.1371/journal.pone.0092126.g002

MAVS Protein Is Attenuated by RV NSP1

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Proteasome inhibitor interferes with MAVS degradationTo determine whether the NSP1-dependent loss of MAVS in

RV-infected cells occurred via the proteasome pathway, HEK293

cells were treated with MG132 (20 mM for 6 h) following co-

transfection of pFLAG-MAVS and pcD-NSP1. As shown in

Figure 3A, MAVS protein expression was significantly restored in

presence of MG132. To further elucidate the mechanism, in vitro

ubiquitination assay was performed where HEK293 cells were

cotransfected with plasmids expressing pFLAG-NSP1 together

with pFLAG-ubiquitin in the presence or absence of proteasome

inhibitor MG132 (20 mM for 6 h), followed by immunoprecipi-

tation using anti-MAVS Ab. Immunoblot analysis using anti-

FLAG Ab revealed that full length NSP1 resulted in ubiquitination

of MAVS. These results indicate that NSP1 has a role in MAVS

ubiquitinylation and its proteasome-mediated degradation.

Rotavirus NSP1 inhibits MAVS mediated interferon betaactivation irrespective of IRF3 degradation

To determine the downstream role of MAVS degradation

mediated by NSP1, we co-expressed pFLAG-MAVS and pcD-

NSP1 constructs in HEK293 cells and qRT-PCR was performed.

In MAVS expressing cells, .300 fold induction of IFN-b and 200

fold induction of ISG56 transcripts was observed (Figure 4A).

However when NSP1 was co-expressed, significant downregula-

tion of both IFN-b and ISG56 was observed in a dose dependent

manner (Figure 4A). To mimic virus induced activation of innate

immune response in cells, the cells were transfected with poly dA

dT which transcribes into dsRNA with a 59-triphosphate moiety

(59pppdsRNA) and is indirectly sensed by RIG-I [1, 18 and 19]. In

HEK293 cells, poly dA dT transfection resulted in significant

increase in IFN-b (.80 fold) and ISG56 (.30 fold) transcript

compared to control (Figure 4B), which was inhibited in presence

of increasing concentration of NSP1 (Figure 4B). Similar results

were obtained with Poly IC, a synthetic dsRNA analogue (data not

shown).

IRF3 and NFkB luciferase assays were performed to study the

functional role of NSP1 in MAVS dependent IRF3 and NFkB

activation. For IRF3 reporter assay, HEK293 cells were

cotransfected with IRF3 luciferase reporter vector, pCMV-MAVS

in presence or absence of pcD-NSP1. For dual luciferase NFkB

reporter assay, HEK293 cells were cotransfected with NFkB–luc

(containing IL8 promoter), pRL-TK, pCMV-MAVS and pcD-

NSP1 or pcDNA. The results revealed more than 50 fold

reduction in both IRF3 and NFkB activation in presence of

NSP1 as evident in Figure 4C. For confirmation, the nuclear

fractions were isolated from poly AT transfected (12h) HEK293

cells expressing either pcD-NSP1 or empty vector control.

Consistent with the qRT-PCR data, in cells expressing NSP1,

significantly less nuclear translocation of IRF3 and NFkB was

observed. The levels of pIkBa were checked in the cytosolic

fraction which revealed lower phosphorylation in presence of

NSP1 (Figure 4D).

As NSP1 is already known to downregulate both IRF-3 and NF-

kB activation [24], it was necessary to assess whether degradation

of MAVS has actually any direct effect on IFN induction. Since

previous report has shown inability of OSU NSP1 to downregulate

IRF3 [31], the NSP1 construct from OSU strain was used.

pFLAG-MAVS vector was co-transfected with pcD-NSP1-OSU

and IRF3 phosphorylation was checked. As shown in Figure 5A,

pcD-NSP1-OSU was able to inhibit IRF3 phosphorylation, all

though there was no degradation of IRF3. When the membranes

were reprobed with MAVS antibody there was loss of MAVS

protein in cells expressing OSU-NSP1. These results were

consistent with our previous results in Figure 2A where MAVS

was degraded even in OSU infected cells. Thus, NSP1 can

modulate IFN induction by MAVS degradation irrespective of its

IRF3 degrading property. Furthermore, TBK1 and IRF3-5D, a

phosphomimetic form of IRF3 were overexpressed, along with

NSP1 in presence and absence of MAVS to analyze the induction

of IFN-b transcripts. As overexpression of TBK1 or IRF3-5D

results in constitutively phosphorylated IRF3, our aim was to

analyze effect of NSP1 on MAVS in presence of constitutively

active IRF3. As shown in Figure 5B, in presence of both NSP1 and

MAVS, the IFN-b levels induced by TBK1 overexpression, were

significantly inhibited compared to only pcD-NSP1 transfected

cells. Similar results were achieved when IRF3 phosphorylation

Figure 3. Proteosome-mediated MAVS degradation. A) NSP1 induced MAVS degradation is prevented in presence of MG132. HEK293 cellswere either transfected with FLAG-MAVS or co transfected with pFLAG-MAVS and pcD-NSP1 in presence or absence of 20 mM MG132 followed byimmunoblot analysis with anti-FLAG, anti-GAPDH and anti-His. B) NSP1 induces ubiquitinylation of MAVS. HEK293 cells were transfected withexpression vector encoding FLAG-tagged ubiquitin with presence or absence of pcD-NSP1. Cells were grown in DMEM containing MG132 (20 mM) for6 h. Anti-MAVS immunoprecipitates were analyzed by immunoblotting His-tagged ubiquitin with anti-His Ab. Whole-cell lysates were subjected toimmunoblotting with anti-His and anti-GAPDH was used as an equal loading control. Figure also showed that MG132 affects MAVS degradation butnot ubiquitinylation.doi:10.1371/journal.pone.0092126.g003

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was measured by western blotting. As shown in Figure 5C, IRF3

phosphorylation was inhibited more in cells overexpressing NSP1

along with MAVS and TBK1 compared to only TBK1.

Comparable results were achieved with overexpression of IRF3-

5D (Figure S2). In addition, interaction between TBK1 and

MAVS was analysed in presence and absence of NSP1 by Co-IP.

As MAVS is reported to interact with TBK1 protein for its

downstream functioning, it was hypothesized that degradation of

MAVS could directly affect its interaction with TBK1. HEK293

cells were transfected with pFLAG-MAVS and pcD-NSP1 in the

presence of MG132. Experiments confirmed reduced interaction

of MAVS-TBK1 in presence of NSP1 (Figure 5D). Thus,

ubiquitinylation of MAVS protein hampers its proper functioning

as an adapter molecule. Overall, results confirmed that even in

presence of activated IRF3 there is considerable downregulation of

IFN response which is due to degradation of MAVS protein

during RV infection.

NSP1 interacts with MAVS proteinRole of NSP1 in MAVS degradation was confirmed, but

whether NSP1 directly interacted with MAVS to cause degrada-

tion or it was an indirect effect was not clear. To analyze, the cells

were infected with SA11 for 0-8 hours and co-immunoprecipita-

tion was done with the NSP1 antibody followed by immunoblot-

ting with MAVS. As shown in Figure 6A, interaction between

NSP1-MAVS was observed as early as 2hpi which gradually

increased until 8 hpi. The interaction of NSP1 with MAVS was

confirmed by reciprocal co-IP experiments as shown in Fgure 6B.

To rule out role of any other rotaviral protein during this

interaction, pcD-NSP1 and FLAG-MAVS were co-transfected in

Figure 4. NSP1 inhibits, MAVS mediated IFN-b induction. A & B) Fold change of IFN-b and ISG 56 were calculated in presence or absence ofNSP1 in MAVS overexpressing, or poly AT stimulated cells. RNA was extracted from transfected HEK293 and qRT-PCR was performed with SYBR greenMastermix. GAPDH was used as reference gene. Data presented as fold change (based on 22DDCt values) relative to mock transfected control cells(mean 6 SD; n = 3). C) Relative increase in IRF3 and NF-kB promoter activity was measured in cells transfected the pLuc-4xIRF3 or pLuc-IL8 luciferasereporter vectors, pRL-TK as loading control in absence or presence of pcD-NSP1. Luciferase activities were determined using the Dual LuciferaseReporter Assay Kit (Promega, Madison, WI). The data are presented as the fold change in luciferase units (mean 6 SD; n = 3, P,0.05) relative to mockcontrol and was normalized with the Renilla luciferase activity. D) Cytosolic and nuclear proteins were isolated from HEK293 cells with ProteoJETCytoplasmic and Nuclear Protein Extraction Kit. Cytoslic fractions were analyzed for pIkBa and nuclear fraction aliquots were immunoblotted withIRF3, NFkB, LaminA/C and actin antibodies.doi:10.1371/journal.pone.0092126.g004

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HEK293 cells. Cells were lysed 24 hours post-transfection, and

CO-IP (Pierce IP kit) was performed with FLAG or His antibodies

targeting MAVS or NSP1 respectively. Immunoblotting with

reciprocal antibodies revealed that there is a significant interaction

between NSP1 and MAVS protein (Figure 6C).

In order to map the domains responsible for this interaction,

deletion mutants of NSP1 and MAVS were constructed. pcD-

NSP1-N100 (N terminal 100 amino acids including the putative

the RING-E3 ubiquitin ligase domain), pcD-NSP1-C395 (C-

terminal 101 395 amino acids) and pcD-NSP1DIRF3BD (NSP1

lacking IRF3 binding domain) (Figure 7A). Two construct were

generated for MAVS, Mini MAVS, comprising of only CARD

domain followed by the TM domain and Pro-MAVS comprising

the remaining proline rich domain. The CARD domain is

responsible for the interaction of MAVS with upstream signaling

proteins and the TM region is responsible for the mitochondrial

translocation. Co- immunoprecipitation experiments carried out

in cells expressing pcD-NSP1-N100 or pcD-NSP1-C395 or pcD-

NSP1DIRF3BD, revealed strong interaction between C- terminal

fragment (NSP1-C395) and MAVS protein (Figure 7B). Both pcD-

NSP1-N100 and pcD-NSP1DIRF3BD did not interact with

MAVS. To correlate Co-IP results with functionality, HEK293

cells were co-transfected with MAVS and either pcD-NSP1-N100

or pcD-NSP1-C395 or pcD-NSP1DIRF3BD and IFN-b and

ISG56 transcripts were analysed by real-time PCR. In spite of

interaction with MAVS, C-395 domain of NSP1 could not inhibit

MAVS induced IFN-b (Figure 7C). Unlike FL-NSP1, pcD-NSP1-

N100 and pcD-NSP1DIRF3BD constructs also could not inhibit

MAVS induced IFN-b (Figure 7C).

In order to map the domain of MAVS responsible for its

interaction with NSP1, Co-IP experiments were carried out in cells

expressing pCD-NSP1 along with pFLAG-CARD-TM or Pro-

MAVS. Co- immunoprecipitation experiment revealed that

similar to full length MAVS, NSP1 can interact with mini MAVS

(Figure 7D). Mini-MAVS has also been shown to induce IFN-bsimilarly to WT MAVS [13]. Thus, in order to assess whether this

Figure 5. NSP1 inhibits IFN-b induction irrespective of IRF3 degradation. A) HEK293 cells were transfected with FLAG-MAVS and pcD-OSU-NSP1 vector in order to assess the MAVS mediated inhibition of IRF3 phosphorylation. Cell lysates were analyzed for pIRF3, IRF3, Anti-His, Anti-FLAGand GAPDH specific antibodies. B) Fold change of IFN-b transcripts was assessed in cells overexpressing human TBK1 and pFLAG-MAVS vectors, inpresence or absence of pcD-NSP1. The data shown are means 6 the SD (n = 3). * Significantly different in comparison to human TBK1 and NSP1transfected and pFLAG-MAVS untransfected condition. P,0.05 C) Activation of IRF3 was assessed in absence or presence of pcD-NSP1 in cellstransfected with TBK1 and FLAG-MAVS. D) Association of MAVS with TBK1 was studied by Co-IP in presence or absence of pcD-NSP1 in cellsoverexpressing TBK1 and MAVS. The MAVS degradation was controlled by proteosomal inhibitor MG132 Results reveal reduced interaction betweenMAVS-TBK1 in presence of NSP1.doi:10.1371/journal.pone.0092126.g005

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interaction has any effect on CARD-TM expression levels,

HEK293 cells were co-transfected with pFLAG-CARD-TM along

with increasing concentration of NSP1. Consistent with previous

results with full length MAVS, NSP1 degraded Mini-MAVS

(CARD-TM) as well as inhibited IFN-b transcript .50 fold

(Figure 7E). Expression of Mini-MAVS and NSP1 in cells were

confirmed by immunoblotting (Figure S3). Overall results suggest

that CARD domain and TM domain of MAVS are sufficient to

interact with NSP1 and inhibit IFN pathway, however, full length

NSP1 is required for degradation of MAVS.

Identification of MAVS aggregates and its inhibition byNSP1

It has been reported that during viral infection MAVS forms

large detergent resistant aggregates which activate IRF3 in cytosol

[5].To analyze kinetics of MAVS aggregate formation during RV

infection and role of NSP1 in this process, SDD-AGE (semide-

naturing detergent agarose gel electrophoresis) was used as

described previously [5]. A fraction of the SDD-AGE lysate was

kept for running SDS-PAGE in order to analyze the expression of

MAVS, phospho-IRF3 and COX IV as internal control. The

NSP1 mutant RV strain A5-16 was used for virus infection, since it

does not degrade cellular MAVS (Figure 2B). As shown in Figure

8A, it was noted that MAVS antibody could barely detect MAVS

in uninfected control and 4 hpi infected cells on SDD-AGE,

whereas MAVS expression was confirmed in the same samples

following regular SDS-PAGE. Thus MAVS aggregates are formed

as early as 4hpi in infected cells which co-relates with downstream

signaling as shown by the phosphorylation of IRF3. In order to

confirm the role of NSP1 on MAVS aggregate formation,

HEK293 cells were transfected with pcD-NSP1 and FLAG-

MAVS protein followed by infection with A5-16 (NSP1 mutant)

strain, in presence of either MG132 or DMSO. Virus infection

was done to induce MAVS aggregation as MAVS overexpression

alone induces very low level of aggregates [5]. Membrane was

immunoblotted with anti-FLAG antibody to avoid detection of

cellular MAVS. In cells overexpressing only MAVS, A5-16

infection resulted in the formation of MAVS aggregates (Figure

8B-lane 2). In cells overexpressing both NSP1 and MAVS, no

aggregates were observed (Figure 8B-lane 3), however in presence

of MG132 aggregates were restored (Figure 8D-lane 4). Thus,

degradation of MAVS by NSP1 not only affects its interaction with

downstream molecules (TBK1) but also inhibit downstream

Figure 6. NSP1 interacts with MAVS protein during infection and transfection. A) NSP1 interacts with MAVS during SA11 infection. HT29cells were infected with SA11 for increasing time points and cell extracts were immunoprecipitated with either NSP1 or MAVS antibody followed byimmunoblotting with reciprocal antibody. Whole cell lysates were immunoblotted with anti-MAVS and anti-NSP1 antibody. CO-IP revealed positiveinteraction between NSP1 and MAVS. B) HEK293 cells were co-transfected with pFLAG-MAVS and pcD-NSP1. After 24 hours, cell extracts wereimmunoprecipitated using FLAG Ab or His Ab followed by immunoblotting by reciprocal antibodies. NSP1 was found to co-immunoprecipitate withMAVS in absence of other viral protein.doi:10.1371/journal.pone.0092126.g006

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aggregate formation on mitochondrial membrane in response to

virus infection.

Discussion

Host cellular response to virus infection involves the concom-

itant activation of parallel signaling pathways leading to the

transcription of a plethora of cytokine genes, prominent among

these are the genes encoding type-I IFN. It is the action of IFNs

that interconnect the innate immune responses into the adaptive

response, which act in an auto-, para- and endocrine manner

designed to defend against viral infection [38]. Several viruses

have evolved sophisticated mechanisms to evade the host innate

immune response by directly interfering with the activation or

downstream signaling events associated with PRR signal propa-

gation [39]. As it is known that MAVS is a potent inducer of IFN-I

responses [6,40–42], therefore it was important to determine

whether RV regulates this adapter protein during virus replication.

Here it is shown that rotaviral NSP1 down regulates the adaptor

protein MAVS by proteosomal degradation thereby creating an

environment devoid of viral RNA sensing machinery.

NSP1 subverts the host innate immune response by inducing

the degradation of factors necessary for IFN-b signaling. Until now

NSP1 is reported to down regulate IRF3, IRF5, IRF7, b-TrCP

and RIG I [23–26,44]. In addition, recently NSP1 was found to

inhibit STAT1 activation [43] and induce NSP1 mediated

degradation of p53 and TRAF2 proteins [29,30]. A detailed study

have been done by Arnold et al on the role of NSP1 from different

virus strains, in targeting IRFs for inhibiting IFN-b [37]. There it

was revealed that NSP1 of RV strains OSU, KU, Wa and DS-1

were unable to degrade IRF3 but were still competent enough to

Figure 7. Domains of NSP1 and MAVS responsible for the interaction. A) Diagrammatic representation of mutants of NSP1 and MAVS usedin the study. For NSP1 there were 3 mutants; pcD-NSP1-100 which comprised the putative ubiquitin ligase domain, pcD-NSP1-C-395 containing theremaining 395 amino acids of C-terminal and pcD-NSP1DIRF3BD comprising the N-terminal 327 amino acids. For MAVS, two constructs weregenerated, one comprising the CARD and TM domain (pFLAG-CARD-TM) and other vector encoding the intermediate proline rich domain of MAVS(pFLAG-Pro-MAVS). B) To map the domain of NSP1 responsible for this interaction, HEK293 cells were co-transfected with the NSP1 mutants andpFLAG-MAVS. Pull down was performed with anti FLAG Ab which revealed a positive interaction between pcD-NSP1-C-395 and MAVS. Membranewas probed with Anti-FLAG Ab. C) Relative fold change over control was measured for IFN-b and ISG 56 gene transcripts in cells co-expressing fulllength NSP1-N-100, NSP1-C-395 and NSP1DIRF3BD with MAVS. The data shown are means 6 the SD (n = 3). * Significantly different in comparison tomock transfected condition. P,0.05.D) To identify the domain of MAVS protein responsible for the interaction, two construct pFLAG-CARD-TM (MiniMAVS) and pFLAG Pro-MAVS were co-transfected with pcD-NSP1 and pulled down with anti-His Ab. CARD-TM domain of MAVS interacts with NSP1.E) Relative fold change in IFN-b gene transcript in cells expressing mini-MAVS in presence or absence of NSP1 with respect to control was measuredby real-time PCR. The data shown are means 6 the SD (n = 3). * Significantly different in comparison to pcD-NSP1 untransfected condition. P,0.05.doi:10.1371/journal.pone.0092126.g007

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inhibit IFN-b response. OSU was found to degrade b-TrCP

whereas KU, Wa and DS-1 targeted IRF5 and IR7. The finding

that MAVS protein is also degraded during rotaviral infection

brings forth the fine-tuning strategies carried out by the virus

under different conditions. Our study shows degradation of

MAVS during infection in a RV strain independent fashion.

Extent of MAVS degradation varies from one RV strain to

another with respect to control but does not co-relate with levels of

IRF3 (Figure 2D). In addition overexpression of NSP1 from OSU,

KU, DS-1 or Wa strains also resulted in degradation of MAVS

irrespective of their ability to degrade IRF3. Therefore the ability

of an NSP1 protein to degrade one target protein does not

necessarily mean that it can also degrade other protein. This

clearly indicates the presence of various complementary inhibition

mechanisms. Thus, MAVS is universally targeted by the RV

strains, during the early steps of infection to escape IFN induced

antiviral signaling.

Since the RV NSP1 of most strains is highly competent in

degrading IRFs it was difficult to devise an assay where the sole

effect of MAVS down regulation on IFN modulation could be

studied. To overcome this OSU-NSP1 was overexpressed as it has

been shown to be defective in IRF3 degradation [37]. Results

confirm no effect on IRF3 in OSU-NSP1 expressing HEK293

cells but MAVS degradation and inhibition of MAVS induced

IRF3 phosphorylation was observed suggesting that both mech-

anisms are independent (Figure 5A). When IFN-b transcripts were

assessed, inhibition of MAVS induced IFN-b was also observed in

presence of OSU-NSP1 (data not shown). This is consistent with

the previous report where inhibition of IFN-b was found in OSU

infected HT29 cells [37]. This was further confirmed when TBK1

was overexpressed along with MAVS and full length NSP1 (SA11).

As expected NSP1 inhibited TBK1 induced IFN-b response to

almost one-third compared to only TBK1. When MAVS was

further co-expressed with TBK1, a huge induction (.250 fold) of

IFN-b was observed which was significantly downregulated

(.80%) in presence of NSP1 (Figure 5B), confirming the

significance of MAVS degradation. Overall it can be postulated

that MAVS degradation is a part of the whole IFN antagonizing

property of NSP1.

NSP1 has a RING domain in the N terminal (1-82 amino acids)

which performs the ubiquitinylation of specific proteins in a

fashion similar to ubiquitin conjugating enzymes (E2). Previous

reports suggest NSP1 induces degradation of IRFs and b-TrCP

proteosomally except RIG-I, which is degraded by a different

unknown mechanism. In our present study MAVS degradation

was also found to be proteosome mediated, as it was rescued in

presence of proteosomal inhibitor, MG132. To know whether

NSP1 binds with MAVS for its degradation, co-immunoprecip-

itation was done both in cells infected with RV strain SA11 as well

as in cell overexpressing FLAG-MAVS and pcD-NSP1. NSP1 was

found to co-immunoprecipitated with MAVS both during viral

infection and overexpression suggesting an interaction in absence

of other viral protein. Deletion mutant of NSP1 suggested that the

C-terminal of NSP1 was sufficient for its interaction with MAVS;

however as the RING domain is in N-terminal, a full length NSP1

was necessary for MAVS degradation. Previous studies by Qin et al

had also reported through luciferase assay that NSP1DIRF3

binding domain is incapable of downregulating MAVS induced

IFN-b. This might be explained, since removing the IRF3-BD

domain of NSP1 hampers its MAVS downregulating property.

Furthermore mutants of MAVS were constructed to identify the

domain responsible for binding with NSP1. MAVS protein

comprises of a typical caspase activation and recruitment domain

(CARD) at its N-terminal followed by a proline-rich region (Pro) in

Figure 8. Formation of MAVS aggregates during Rotavirus infection. A) Crude mitochondrial extracts were prepared from HEK293 cellsinfected with A5-16 strain (3 M.O.I.) at increasing time points (4, 8 and 12). Extracts were analyzed by SDD-AGE to assess the MAVS aggregateformation. Results revealed MAVS aggregate formation from 4 hpi. B) Role of NSP1 on MAVS aggregation and ubiquitinylation was observed byoverexpressing FLAG-MAVS in absence or presence of NSP1. Infection of A5-16 was used for inducing MAVS aggregation, as overexpression of MAVSalone is insufficient for inducing aggregate formation. Results show inhibition of MAVS aggregates in presence of NSP1, which gets restoredfollowing MG132 (20 mM) treatment. A fraction of SDD-AGE lysate was analyzed by SDS-PAGE followed by immunoblotting of MAVS, p-IRF3 and COXIV.doi:10.1371/journal.pone.0092126.g008

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the middle and a hydrophobic transmembrane (TM) domain at

the C terminus. Co-IP studies revealed that CARD-TM domain

was necessary for binding with NSP1 whereas Pro region did not

interact with NSP1 (Figure 4D). In addition to binding with NSP1,

CARD-TM domain alone could inhibit IFN-b transcription in

presence of NSP1 similar to full length MAVS.

Recently it was shown by Hou et al [5] that in response to viral

infection, RIG-I-like RNA helicases bind to viral RNA and

activate the mitochondrial protein MAVS. This activation leads to

the formation of very large MAVS aggregates on the mitochon-

drial membrane which interacts with cytosolic signaling proteins,

such as TRAFs, resulting in the activation of IKKs and TBK1.

These MAVS aggregates were detergent resistant in nature and

therefore a method called semidenaturing detergent agarose gel

electrophoresis (SDD-AGE) was employed, which was previously

used for the detection of prion-like structures. Since A5-16 (NSP1

mutant), strain did not degrade MAVS; MAVS aggregates were

studied followed A5-16 infection. A smear of SDS- resistant high

molecular weight MAVS was observed in time dependent manner

during RV infection (Figure 8A). No change in overall MAVS

expression was observed by immunoblotting. Similar results were

obtained when HEK293 cells were transiently transfected with

FLAG-MAVS and infected with A5-16. MAVS aggregates were

abrogated in presence of NSP1 but were restored when MG132

was added. Restoration of MAVS aggregates in presence of

MG132 further confirms that MAVS degradation by NSP1 is

proteasome mediated.

In most mammalian cells, activation of IRF3 leads to the

expression of IFN-b, which in turn induces the expression of

master regulator IRF7. By targeting IRF3 for degradation, NSP1

removes an upstream transcription factor needed for IRF7

expression, thereby subverting the production of type I IFN.

The dual mechanism of NSP1 in subverting IRF7 function may be

crucial for successful virus replication in the host at later stages of

infection, where the virus may be challenged in cells that have

transitioned from a naive to an antiviral status due to exposure to

cytokines or debris from neighboring infected cells. However, by

degrading MAVS, RV may escape the initial antiviral defense

mechanisms activated by the RIGI–MDA5 pathway. During RV

infection there is a potential redundancy in the functions of RIG-I

and MDA-5 [18], therefore abrogating MAVS results in a

complete shutdown of the host RNA sensory machinery.

Based on the results obtained in this study as well as previous

reports, we propose a model for the NSP1 mediated modulation of

Figure 9. Mechanistic model for rotavirus NSP1-mediated attenuation of cellular proteins for improved infection. The modelsummarizes all the NSP1 mediated inhibitory effects during RV infection. In brief, it shows the critical role of MAVS in RLR mediated activation of typeI IFN and immune response. By degrading MAVS, NSP1 can directly inhibit IRF3 and NF-kB signaling. Other cellular proteins like IRFs and b-TrCP aretargeted selectively but MAVS degradation was observed in all RV strains with functional NSP1.doi:10.1371/journal.pone.0092126.g009

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RV innate immune response (Figure 9). Briefly, RV cell entry and

subsequent replication leads to MDA-5 and RIG-I activation

which may be activated by different types of viral PAMPs [18].

Initially the adaptor MAVS associates with activated PRRs and

propagates signaling to activate IRF3 and NF-kB. But during RV

infection, expression of NSP1 leads to the degradation of RIGI,

MAVS, IRF3 and IRF7 and thus IFN-b secretion. For most of the

animal RV strains where NSP1 effectively degrades IRF3, the

primary antiviral response is inhibited significantly which is further

accentuated by degradation of MAVS protein. In case of RV

strains, such as OSU, Wa, DS-1 or KU which cannot efficiently

degrade IRF3, down regulation of MAVS results in a sustained

inhibition of IFN-b secretion to maintain pro-viral state in cell.

Overall, all targets of NSP1 directly or individually inhibit IFN

activation, suggesting multiple strategies to evade IRF-dependent

and -independent signaling pathways. Further understanding of

these mechanisms should yield novel strategies for developing

antivirals that evoke responses to eliminate RV infection.

Supporting Information

Figure S1 The inhibition of viral replication induced byUV treatment. To prepare UV-inactivated RV, simian SA11

were pretreated with 40 mg/ml psoralen AMT and then irradiated

by long-wave UV-light (365 nm) for 2 hours. HT29 cells were

infected with SA11 or UV-SA11at 1 M.O.I. for indicated time

points. (A) RNA was isolated at specific intervals followed by

quantification of nsp4 and gapdh mRNA transcripts by qRT-PCR.

Fold changes were obtained by normalizing relative gene

expressions to gapdh using the formula 22DDCT

(DDCT =DCTSample-DCTUntreated control). (B) At indicated time

points, HT29 cells infected with normal and UV irradiated virus

were freeze-thawed. Extracted and purified viral preparations

were titrated by plaque assay.

(TIF)

Figure S2 Effect of MAVS degradation induced by NSP1on IRF3-5D overexpression. With an aim to analyze the effect

of NSP1 on MAVS during constitutively phosphorylated IRF3,

vector encoding IRF3-5D (a phosphomimetic form of IRF3) was

overexpressed, along with NSP1 in presence and absence of

MAVS followed by immunoblotting with p-IRF3 antibody In

presence of both NSP1 and MAVS, the p-IRF3 levels induced by

IRF3-5D overexpression, were significantly inhibited compared to

only MAVS and IRF3-5D transfected cells. Membranes were

reprobed with IRF3 and GAPDH antibodies.

(TIF)

Figure S3 NSP1 mediated degradation of Mini-MAVS(pFLAG-CARD-TM). In order to co-relate the interaction

between CARD-TM and NSP1, HEK293 cells were transfected

with vectors encoding mini-MAVS and increasing concentration

of pcD-NSP1 for 24 hours. Results reveal dose-dependent

degradation of CARD-TM. Membranes were probed with anti-

FLAG, anti-His and to confirm equal loading GAPDH antibodies.

(TIF)

Acknowledgments

S. Nandi (SRF), P. Bagchi (SRF) and R. Bhowmick (SRF) are supported by

fellowships from CSIR, Govt. Of India and S. Chanda (JRF) is supported

by UGC, Govt. of India.

Author Contributions

Conceived and designed the experiments: SN MCS. Performed the

experiments: SN SC MKN RB. Analyzed the data: SN PB MCS.

Contributed reagents/materials/analysis tools: MCS. Wrote the manu-

script: SN MCS.

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